Steroid conjugates, preparation thereof and the use thereof

ABSTRACT

Conjugates comprising one or more steroids conjugated with one or more mammalian proteins are disclosed. The conjugates are useful for diagnosis or treatment of solid cancer aid haematological malignancies. Further the conjugates exhibits a synergistic action together with a cytoskeleton acting drug such as Taxol®, which enable the treatment of cancers that otherwise would be non responsive to Taxol®.

The present invention relates to new steroid conjugates that are usefulfor the diagnosis and treatment of solid cancers and hematologicalmalignancies.

Further the invention relates to combinations of said steroid conjugateswith cytotoxic agents showing synergistic effects in the diagnosis andtreatment of cancer.

BACKGROUND FOR THE INVENTION

Classical steroid hormone action is mediated through intracellularsteroid hormone receptors. These proteins dimerize, after steroidbinding, translocate to the nucleus, and exert specific nucleartranscription factor effects on specific steroid-sensitive genes [1]. Inrecent years, however, a number of studies indicate that, in addition tothe above genomic action, steroids bear non-genomic effects, mediated inminutes, and implicating different pathways than those involved inclassical steroid receptor action [2,3]. Non-genomic steroid actionswere, in addition, been found in cells not expressing classical steroidreceptors. The above, non-genomic steroid receptor actions wereattributed to another class of steroid receptors, found on membrane ofcells, and being biochemically, immunologically and pharmacologicallydifferent from classical steroid receptors. Until now, non-genomicsteroid effects were found for estradiol, cortisol, and testosterone, inanimal tissues, usually not-expressing classical receptors [2-13].Activation of these non-classical steroid sites, found on membranes ofcells was the increase of the flux of extracellular calcium to thecytosol [8-10, 12, 14], and in some times, modifications of thecytoskeleton [7, 15]. In all cases, BSA-conjugated steroids were used asligands for these extracellular (membrane) steroid sites, in order toidentify these sites. Indeed, covalent binding of steroids with high (60kD) molecular weight proteins makes these molecules to loose their lipidsolubility (and therefore the property of translocating to the cellthrough the plasma membrane) and confines them with water solubility andthe possibility of binding to specific steroid sites. Commerciallyavailable sources of these compounds are currently available (e,g, SigmaChemical Co. St Louis, Mo., USA). Nevertheless, human applications ofthis membrane steroid receptor activation have not been described sofar.

GB 2 068 973 A, disclose conjugates of a steroid and an immunogenicprotein such as human serum albumin for use in compositions forincreasing ovulation in cattle.

EP 1 104 677 A2 discloses conjugates of a protein and a low molecularweight compound, where steroids are mentioned as examples of such.Bovine serum albumin is mentioned as an example of a protein useable insuch conjugates. If at least part of the low molecular compounds iscytostaticum and the protein is a tumor specific antibody, an enzyme ora lectin the conjugates may be used for treatment of cancer.

U.S. Pat. No. 6,372,712 B1 discloses synthetic bifunctional moleculescontaining a drug moiety and a presenter protein ligand. Steroids arementioned as examples of a drug moiety and albumin is mentioned as apreferred example of a presenter protein. The conjugates may be used toenhance the binding affinity and/or specificity of the agent to itstarget. It is stated that the conjugates can be used for treatment ofdifferent diseases dependent on the type of drug moiety used, but thereare no specific teachings on which diseases may be treated with whichdrug moiety.

WO 01/82910 A2 discloses therapy of cancer using a compositioncomprising progesterone and RU486 (Mifesterone) or derivatives thereofand a portion of HPV E2 protein for the treatment of cervical cancer orcertain pre-cancerous cervical lesions. The HPV E2 protein is known tobe lethal for the cervical tissue and the connection with the steroidseems to enhance this effect. The steroid may be bound to a steroidcarrier protein such as human serum albumin.

U.S. Pat. No. 4,215,102 A1 discloses a conjugate consisting ofprogesterone or estrogen, protein and a fluorochrome. The conjugates maybe used for detection of steroid hormone receptors in excised humantissue sections.

WO 99/13914 A1 disclose the pharmaceutical compositions comprisingtherapeutic active substances having low aqueous solubility bound to aplasma protein fraction in controlled aggregation state in order toincrease the soluble amount of the active substances and therebyincrease the availability thereof.

WO 93/02691 A1 disclose a delivering system for glucocorticoids using aprotein carrier molecule having a binding site for glucocorticoids and abinding site for the targeted cell population. There is not indicatedparticular indication for which pharmaceutical compositions comprisingsaid delivering systems are useful but it may be used for conditionsknown to be treatable with glucocorticoids.

DESCRIPTION OF THE INVENTION

The invention is based on the discovery that steroid conjugated to amammalian protein may surprisingly have a cytotoxic effect on cancercells, even when neither the steroid nor the mammalian protein by itselfexert any substantial cytotoxic effect.

Thus in one aspect the invention related to the use of one or moresteroids thereof, which are not cytostaticum, conjugated with mammalianproteins for the manufacture of pharmaceutical compositions for thetreatment of solid cancers or hematological malignancies.

The steroid may in principle be any steroid thereof having a cytostaticeffect when conjugated to a soluble mammalian protein. The term“steroids” is according to the invention intended to comprise allnatural and synthetic steroid hormones, their analogs and derivativesthereof such as sulphate and fatty acid esters, their precursors,metabolites and their analogs, which may be steroidal or not steroidalin structure.

As analogs the inventors contemplate all natural, semisynthetic orsynthetic polycyclic molecules, capable to bind to human membranesteroid receptors, their mixtures, precursors and metabolites.

In one preferred embodiment the steroid is a steroid capable of bindingto a membrane associated steroid receptor.

As examples of suitable steroid according to the invention can bementioned: glucocorticoids, cortisol, testoterone, estrogen, estradiol,progesterone and any known analogs thereof.

The mammalian protein may according to the invention in principle be anymammalian protein that is water soluble when conjugated to steroid.

The mammalian protein may be selected among globular proteins, plasmaproteins, albumins, binders or antibodies of selective human tumoralcell antigens.

Albumins are preferred examples of proteins to be conjugated to steroidsaccording to the invention.

Human albumins and bovine serum albumin (BSA) are examples of preferredproteins to be conjugated according to the invention.

Human albumins are particular preferred in case that the particularconjugate is intended for treatment of a human being.

The term protein is contemplated to comprise natural and non naturalproteins. In this context non natural proteins are considered anyprotein that differs from a natural occurring protein in amino acidsequence, glycosylation pattern or chemical modifications. Non naturalproteins may be provided using recombinant DNA technologies or bychemical modifications of natural proteins.

Non natural proteins provide the possibilities of modifying naturalproteins in order to e.g. reduce the antigenicity in a intended host;insert a suitable hapten; increase the stability of the protein againste.g. oxidation; provide a suitable site at the protein for improvedattachment of a steroid; provide a suitable way of producing theintended protein.

Non natural proteins are considered mammalian protein if they directlyor indirectly are derived from natural mammalian proteins, such as bye.g. chemical modification of isolated natural protein, or byrecombinant DNA technologies, where a gene encoding a mammalian proteinis used in is original or in a modified form.

Natural proteins may be provided from a natural source or from tissuecultures of natural or recombinant cells provided by recombinant DNAtechnologies.

It is within the skills of the average practioner to provide a givensuitable natural or non natural protein using known procedures.

If the steroid protein conjugates according to the invention is intendedfor use in a treatment regimen the protein is preferably selected sothat it is not immunogenic in the intended subject for the treatment.

The conjugates according to the invention may be prepared by proceduresthat are known per se, wherein the steroid is attached to the protein.The attachment may be any attachment that provides for a stableconjugate, preferably a covalent attachment.

In addition to steroid further groups may be attached to the proteine.g. a labelling attached for diagnostic purposes e.g. a hapten, acoloured moiety, a fluorescent moiety, a radionuclid etc.

The covalent attachment of the steroid moiety to proteins will be madeby the use of conventional methods (ex. attachment of a carboxy-methylether moiety, and the attachment to proteins by the action ofcarboxydiimide).

In order to increase the selectivity of the steroid-protein conjugates,different attachments of the steroid moiety to the protein will be made(ex. attachment trough addition of an acid group at carbon positions 1,3, 7, 11 or 15 of the steroid—the list is not exclusive-).

According to the invention the conjugates may be used for treatment ofsolid cancers and haematological malignancies.

As examples of solid cancers can be mentioned: prostate adenocarcinoma(hormone sensitive and resistant) and its metastases (lymph node, boneetc.), breast cancer (hormone sensitive and resistant) and itsmetastases in any places (lymph node, bone etc.), phenochromocytomas andtheir metastases, bone tumors and their metastases brain tumors(neuroblastomas) etc. (the list is not exclusive).

As examples of haematological malignancies can be mentioned: Accute andchronic myeloid leukaemia, acute and chronic lymphoid leukaemia andlymphomas (B and T).

In principle the conjugates may be used for the treatment of thementioned indications in any mammal in need for such a treatment.

As examples of mammals that may be treated using the conjugatesaccording to the invention can be mentioned: human beings, cattle, dog,sheep, horse, goat, donkey, cat and monkeys. Preferably the conjugatesare used for the treatment of humans.

In a preferred embodiment the conjugates according to the invention isused for the treatment of cancer or haematological malignancies in ahuman being.

For such treatment the conjugates may in principle be administered inany known administration form such as oral or rectal administration orby parenteral, percutaneous or intravenous injection, where parenteral,percutaneous or intravenous injection are preferred.

The dosages and regimens are generally to be determined in accordance tothe discretion of the attending physician, taking due considerations tothe patient's age, weight, condition etc.

Generally the daily dosages may be in the range of 1 mg/kg body weightto 100 mg/kg body weight, preferably in the range of 5 mg/kg body weightto 100 mg/kg body weight, more preferred in the range of 5 mg/kg bodyweight to 50 mg/kg body weight and most preferred in the range of 5mg/kg body weight to 20 mg/kg body weight, and in a particular preferredembodiment the daily dosage is around 7 to 10 mg/kg body weight. Thetreatment is generally continued for up to 6 months, preferably in therange of 2 weeks to 6 months, more preferred in the range of 2 weeks to3 months.

The pharmaceutical compositions may be administered at regular intervalsduring the period of treatment in order to maintain a satisfactoryconcentration of the active compound in the circulation, as the skilledperson will appreciate. Thus the pharmaceutical composition according tothe invention may be administered once or more times daily or even withregular intervals of one or more day e.g. every second day, according tothe discretion of the attending physician taking due consideration tothe treatment efficiency and the acceptance of the patient beingtreated.

Another aspect of the invention might take account the possibledegradation of the conjugate according to the invention and theresulting liberation of free hormone.

In order to obtain this tumors bearing testosterone receptors, anantiandrogen may be added and the tumor treated using a steroidconjugate according to the invention where the conjugate is atestosterone protein conjugate.

In addition, for tumors bearing estrogen or progesterone receptors anantiestroge or an anti progestin respectively will replace theantitestosterone, while protein conjugated estrogens or progestins willreplace testosterone protein conjugates.

In the same line of development, in any tissue in which membrane steroidreceptors will be found the corresponding steroid conjugate with 10times higher antisteroid agent and anticytoskeleton agents may beadministered. It is within the skills of the average practioner todetermine the optimal combination in a given situation using commonskills and routine experimentation.

Thus in the aspect the pharmaceutical composition according to theinvention may further comprise one or more antiandrogens in the casethae the steroid present in the administered conjugate is an androgen.Without wishing to be bound by any particular theory it is believed thatthe antiandrogens block classical androgen receptors, and therebyabolished the effect that hormones liberated from a conjugate mighthave.

Antiandrogens may be added in an amount up to 10 times higher amountthan the amount of steroids in the conjugate administered, calculated ona molar basis.

In case that the steroid present on the steroid-protein conjugate is notan androgen but is estrogen or progesterone the antiandrogene may bereplaced by an antiestrogen or an antiprogestine, respectively.

Antiandrogenes, antiestrogenes and antiprogestines will be known for theperson skilled in the art.

As examples of antiandrogens can be mentioned Cyprotone acetate andflutamide. As examples of anti-estrogens and anti progestines can bementioned tamoxifene and RU486, respectively.

In another aspect the conjugates according to the invention is used fordiagnostic purposes.

For the diagnostic use the specimen is contacted with the conjugatefollowed by detection of bound conjugate by any suitable technique.

In one embodiment the conjugate is used for diagnosis in vivo combinedwith an antiandrogen in order to achieve a pharmaceutical orchecthomy,and block any deleterious effect of free testosterone.

In this embodiment the conjugate is administered to the subject followedby detection of the binding of the conjugate to the target. For such anin vivo diagnostic use, a labelling is preferably attached to theconjugate e.g. a radionuclid or an electrondense compound functioning asa contrast agent for X-ray analysis.

In another embodiment the conjugate is used for diagnosis ex vivo. Inprinciple any biological specimen may be tested for binding of conjugateto the specimen. For such application it may be advantageous if theconjugate is easy detectable, e.g. due to attachment of detectablegroups or by using antibodies specific for the protein part of theconjugate.

The inventors have further found a synergy between the conjugateaccording to the invention and a cytoskeleton acting drug.

Thus in s further aspect the invention relates to a pharmaceuticalcomposition comprising one or more steroids, which are not cytostaticum,conjugated with a mammalian protein and a cytoskeleton-acting drug.

The surprising synergy between the conjugate according to the inventionand cytoskeleton acting drugs allow the treatment of solid cancers andhaematological malignancies, which otherwise would be non responsive oronly low or moderate responsive to cytoskeleton acting drugs, using thiscombination.

Thus in a further aspect the invention relates to the use of apharmaceutical composition comprising one or more steroids, which arenot cytostaticum, conjugated with a mammalian protein and acytoskeleton-acting drug for the treatment of solid cancer orhaematological malignancies.

The term “cytoskeleton acting drugs” is according to the invention usedin the usual meaning. As examples of a cytoskeleton-acting drug can bementioned Taxol® or Taxotere®.

The treatment using a combination of the conjugate according to theinvention and a cytoskeleton-acting drug may be performed byadministration of one pharmaceutical composition comprising both activecompounds, or it may be performed by administration of separatepharmaceutical entities, one comprising the conjugate and anothercomprising the cytoskeleton acting drug.

The dosages and regimens of the conjugate and the cytoskeleton-actingdrug are similar to the corresponding dosages and regimens applied ifthe particular conjugate and drug were administered separately.

If the conjugate and the cytoskeleton-acting drug and/or the antisteroidcompound are administered as separate pharmaceutical compositions, theymay conveniently be provided in a kit comprising these twopharmaceutical compositions.

Thus in a further embodiment the invention relates to a kit comprising:

-   -   a pharmaceutical composition comprising one or more steroids,        which are not cytostaticum, conjugated with a mammalian protein;        and    -   a pharmaceutical composition comprising a cytoskeleton acting        drug, or    -   one of an antiandrogen, an antiestrogen and an antiprogestin in        case that the tumor is bearing testosteron-, estrogen- or        progesterone-receptors, respectively.

In an even further embodiment the invention relates to a kit comprising:

-   -   a pharmaceutical composition comprising one or more steroids,        which are not cytostaticum, conjugated with a mammalian protein;        and    -   a pharmaceutical composition comprising a cytoskeleton acting        drug, and    -   one of an antiandrogen, an antiestrogen and an antiprogestin in        case that the tumor is bearing testosteron-, estrogen- or        progesterone-receptors, respectively.

The invention consists of the determination, production and use ofmembrane steroid receptors agonists, determined by the associationbinding of these molecules with membrane steroid receptor bearing cells.

The invention will use these molecules for the diagnosis and treatmentof solid tumor and hematologic malignancies in humans.

The specific molecules will be used, according to the invention, for theproduction of diagnostic and therapeutic agents. They areprotein-conjugated (BSA-conjugated, Human Serum Albumin(HSA)-conjugated, binders or antibodies of selective human tumoral cellantigens for example—the list is not exclusive-) steroids.

The invention will be used, as illustrated in the examples provided, toproduce specific diagnostics in cases of solid tumors and hematologicalmalignancies.

The action of membrane steroid receptors, as illustrated by theexamples, being the modifications of actin cytoskeleton, and thepotentiation and extension of the action of cytoskeleton-acting drugs(eg. Taxol®) makes membrane steroid receptor agonists an interestingclass of potential drugs.

The inventors will therefore target the production of new drugs, capablefor a specific and selective binding to a class of membrane steroidreceptors, present, as illustrated in the provided examples, inselective malignancies, in view of a selective primary or adjuvantchemotherapy. In another aspect, these agents, used aschemotherapeutics, could be used, alone, in combination with antisteroiddrugs or in association with other chemotherapeutics (ex. Taxol® orequivalent drugs), in order to prevent, or modulate the chemoresistanceof selective tumors.

The inventors will determine the best mode of administration of thesedrugs (local or general, injectable or locally applied duringinterventions, etc).

The invention will now be further illustrated in more details in thefollowing examples with reference to the enclosed figures. Othercharacteristics and advantages of the invention are given in thefollowing examples, the references, which are hereby enclosed byreference, and the attached Figures.

It should be understood that the examples and figures are provided forillustration and should not be considered as limiting in any way.

DESCRIPTION OF THE FIGURES

FIG. 1 presents the binding and selectivity characteristics of membranetestosterone receptors in LNCaP cells.

FIG. 2 shows the detection of membrane testosterone receptors, in LNCaPcells by flow cytometry (left panel) and confocal laser scanningmicroscopy (right panel).

FIG. 3 shows the detection of testosterone membrane receptors (by flowcytometry) in cases of prostate cancer, benigh prostate hyperplasia(BPH) and peritumoral non-tumor cells.

FIG. 4 presents the detection of membrane testosterone receptors intouch preparations of prostate tumors (prostate cancer at the left andBPH at right) and histological slides of prostate intraepithelialneoplasia (PIN, at left) and prostate cancer (right).

FIG. 5 presents the detection of estrogen, progesterone and androgenreceptors in ER positive (upper lane) and ER negative breast cancer.

FIG. 6 shows the detection of testosterone, estrogen and progesteronereceptors in bone marrow CD34 and AC133 cells (upper and lower lanes).

FIG. 7 shows the modification of actin cytoskeleton in LNCaP cells bytestosterone-BSA, assayed by confocal scanning laser microscopy (upperpanels) and the increase of polymerized actin by testosterone-BSA,assayed by biochemical methods (lower panel).

FIG. 8 shows the modification of cell viability by a24 hour incubationof testosterone-BSA, alone or associated with Taxol® (A), and the effectafter an additional incubation of 48 hours, in the absence of drugs (B).Panel C presents the dose-response of cells to testosterone-BSA alone oradditioned with Taxol®.

FIG. 9 presents the effect of testosterone-BSA administration in nudemice, bearing implanted tumors. Arrows show the injection times oftestosterone-BSA (500 μl of a 5×10⁻⁶ M solution) with or withoutaddition of 10 mg/kg Taxol®.

EXAMPLES Material and Methods Cell Line

The human prostate cancer LNCaP cell line, originally isolated from alymph node metastasis of prostate adenocarcinoma [16], was purchasedfrom DSMZ (Braunschweig, Germany). Cells were cultured in RPMI 1640medium supplemented with 10% heat inactivated fetal bovine serum (FBS)at 37° C. in a humidified atmosphere of 5% CO₂ in air. They weresubcultured once a week and incubated in serum-free medium for 24 hbefore any experiment. All culture media were purchased from Gibco BRL(Life Technologies, Paisley, UK).

Cell number was assayed using the tetrazolium salt assay [17]. Cellswere incubated for 3 h at 37° C. with the tetrazolium salt (3-(4,5dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, Sigma, St Louis,Mo.). Living cells reduced the dye to purple formazan seen as dark bluecrystals. At the end of the incubation period they were dissolved withpropanol-1 and the absorbance was measure at 575 nm, within one hour.

Detection of Membrane Androgen Receptors

i. Binding Assays

Membrane Preparation

Cells, cultured in 150 cm² flasks without serum, were washed twice withphosphate-buffered saline (PBS), removed by scraping and centrifuged at1500 rpm. Pelleted cells were homogenized by sonication in 50 mMTris-HCl buffer pH 7.4 containing freshly added protease inhibitors (10μg/ml PMSF and 1 μg/ml aprotinin). Unbroken cells were removed bycentrifugation at 2500 g for 15 min. Membranes were obtained bycentrifugation at 45,000 g for 1 hour, and washed once by the samebuffer. Protein concentration was measured by the method of Bradford[18].

Binding Conditions

Saturation binding experiments were performed in a final volume of 0.1ml, containing cell membranes at a final protein concentration of 2mg/ml and at least 6 different concentrations of [³H] testosterone(ranging 2-50 nM) without (total binding) or with (non-specific binding)a 1000-fold molar excess of unlabelled androgen (DHT). For displacementbinding experiments, cell membrane preparations at a final concentrationof 2 mg/ml were incubated with 5 nM of [³H] testosterone (specificactivity 95 Ci/mmole, Amersham-Pharmacia, Buckinghamshire, UK) in theabsence or in the presence of different concentrations of un unlabelledsteroid (DHT, estradiol, progesterone, all from Sigma, St Louis, Mo.),ranging from 10⁻¹² to 10⁻⁶ M. Non specific binding was estimated in thepresence of 5 μM DHT. In both types of binding experiments, after anovernight incubation at 4° C., bound radioactivity was separated byfiltration under reduced pressure through GF/B filters previously soakedin 0.5% polyethylenimine (PEI) in water and rinsed three times withice-cold Tris-HCl buffer. Filters were mixed with 4 ml scintillationcocktail and the bound radioactivity was counted in a scintillationcounter (Tricarb, Series 4000, Packard) with 60% efficiency for Tritium.

ii. Flow Cytometry

LNCaP cells, cultured in serum free medium for 24 hrs, were detachedfrom the culture flask by scraping and suspended in PBS at a density of10⁶ cells/ml. They were incubated at room temperature with 10⁻⁷ Mtestosterone-BSA-FITC conjugate for different periods of time (1 min to1 hour). A thousand-fold BSA-FITC was used to determine non-specificbinding. Cells were analysed by flow cytometry using a Coulter EpicsL-MCL apparatus (Beckman-Coulter Inc. Foullerton Calif., USA) in asample size of 10,000 cells gated on the basis of forward and sidescatter. Testosterone3-(Ocarboxymethyl)oxime-BSA-FITC (namedtestosterone-BSA-FITC), testosterone3-(O-carboxymethyl)oxime-BSA (namedtestosterone-BSA), estradiol6-(Ocarboxymethyl)oxime-BSA-FITC (namedestradiol-BSA-FITC), progesterone3-(O-carboxymethyl)oxime-BSA-FITC(named progesterone-BSA-FITC) and BSA-FITC were obtained from Sigma (StLouis, Mo.).

iii. Confocal Laser Microscopy

LNCaP cells were allowed to grow on poly-L-lysine coated glasscoverslips for at least 48 hours before culture medium was replaced withserum free medium. After a 24-hour period, cells were washed twice withPBS and incubated with Testosterone-BSA-FITC for 30 min in the presenceor in the absence of DHT. As a negative control BSA-FITC was used. Cellswere then washed twice with PBS and fixed with 2% PFA in PBS for 30 min.Coverslips were mounted on to slides using a 1:1 (v/v) mixture ofglycerol and Vestashield (Vector, Burlingame, Calif.). Specimens wereanalysed using a confocal laser scanning microscope (CLSM) (LeicaTCS-NT, Lasertechnik, Heidelberg, Germany).

Detection of Membrane Steroid Receptors in Paraffin-Embedden TissuePreparations

slides were prepared from paraffin blocks of formalin fixed tissuepreparations. Three-four micron (μm) thick tissue sections were cut andput on on SuperFrost Plus slides (Kindler O GmbH, Freiburg, Germany),incubated at 56° C. for 2 h, washed six times with xylene (5 min each),followed by 96%, 80% and 70% ethanol for five minutes each, and finallywith distilled water for 20 min. Tissue slides were then incubated incitrate buffer in a microwaves oven at 500 Watts, three times for 4.5minutes each. Alternatively, slides were incubated at 40 C overnight toremove paraffin in a milder way. Then, they were washed in distilledwater and Tris buffered saline (TBS, 10 mM, pH 7.4). Non-specificabsorption of BSA was eliminated by a 10 min incubation with a 2%solution of BSA in TBS, followed by two washes with TBS. Slides werethen incubated for 10 min with BSA-FITC conjugated steroids and washedwith TBS. Coverslips were mounted on to slides using a 1/1 (v/v) mixtureof glycerol and Vestashield (Vector, Burlingame, Calif.) Specimens wereanalysed using a confocal laser scanning microscope (CLSM) (LeicaTCS-NT, Lasertechnik, Heidelberg, Germany).

Determination of Monomeric and Polymerized Actin

For measurements of the monomeric (Triton soluble) and polymerized(Triton insoluble) actin, LNCaP cells were incubated for 10 min with orwithout DHT or testosterone-BSA (10⁻⁷ M). Then, 500 μl ofTriton-extraction buffer (0.3% TritonX-100, 5 mM Tris, pH 7.4, 2 mMEGTA, 300 mM sucrose, 2 μM phalloidin, 1 mM PMSF, 10 μg/ml leupeptin, 20μg/ml aprotinin, 1 mM sodium orthovanadate, and 50 mM NaF) were added,and the mixture was incubated for 5 minutes on ice. After removing thebuffer, soluble proteins were precipitated with equal volumes of 6% PCA.The Triton-insoluble fraction remaining on the plate was precipitatedwith 1 ml of 3% PCA. Equal volumes of each fraction were subjected toSDS-polyacrylamide gel electrophoresis (SDS-PAGE). The resultingprotein-bands were transferred onto nitrocellulose membrane, and themembrane was blocked with 5% nonfat dry milk in TBS-T (20 mM Tris pH7.6, 137 mM NaCl, 0.05% Tween-20) for 1 h at room temperature. Antibodysolutions (in TBS-T) were added for 1 h at room temperature [monoclonalmouse anti-actin first antibody (Amersham-Pharmacia, Bukinghamshire, UK)and second horseradish peroxidase-coupled antibody (Chemicon, Temecula,Calif.)]. Blots were developed using the ECL system (Amersham-Pharmacia,Bukinghamshire, UK) and the band intensities were quantified by PC-basedimage analysis (Image Analysis Inc., Ontario, Canada) [19].

Immunoprecipitation, Kinase Assays and Immunoblotting Analysis

Testosterone-BSA or DHT-treated, as well as untreated (control) cellswere washed three times with ice-cold PBS and suspended in cold lysisbuffer containing 1% Nonidet P-40, 20 mM Tris pH 7.4 and 137 mM NaCl,supplemented with protease and phosphatase inhibitors. Cleared lysateswere preadsorbed with protein A-Sepharose for 1 h at 4° C., centrifugedand the supernatants (equal amounts of protein) were subjected toimmunoprecipitation using the indicated antibodies and theprotein-A-Sepharose beads.

The lipid kinase activity of PI-3 kinase was measured by the method ofAuger et al [20] with minor modifications. Protein A-Sepharose beadscontaining immunoprecipitated phosphotyrosine proteins were washed threetimes with Buffer A (20 mM Tris pH 7.4, 137 mM NaCl, 1 mM CaCl₂, 1 mMMgCl₂, 1% Nonidet P-40, 0.1 mM Na₃VO₄), three times with 5 mM LiCl in0.1 M Tris (pH 7.4) and twice with TNE (10 mM Tris pH 7.4, 150 mM NaCl,5 mM EDTA, 0.1 mM Na₃VO₄). The immunoprecipitates were then resuspendedin TNE and the PI-3 kinase activity was assayed using 0.2 mg/mlphosphatidylinositol-4,5-bisphosphate (P1-4,5-P2) as a substrate, in thepresence of 58 M ATP, 10 Ci of [-³²P]ATP (5000 Ci/mmol) and 14 mM MgCl₂,for 10 min at 37° C. The reaction was stopped by the addition of 1 M HCland methanol/chloroform (1/1). After mixing vigorously and centrifugingto separate the phases, the lipids in the organic lower phase wereseparated by TLC on oxalated silica gel 60 sheets, as described [21].Chromatographed lipids were also visualized by iodine staining andcompared to the migration of known standards.

For immunoblot analysis, the cell lysates or the immunoprecipitates weresuspended in Laemmli's sample buffer and separated by SDS-PAGE. Proteinswere transferred onto nitrocellulose membrane, and the membrane wasblocked with 5% nonfat dry milk in TBS-T (20 mM Tris pH 7.6, 137 mMNaCl, 0.05% Tween-20) for 1 h at room temperature. Antibody solutions(in TBS-T containing 5% nonfat dry milk) were added overnight at 4° C.(first antibody) and for 1 h (second horseradish peroxidase-coupledantibody). Blots were developed using the ECL system and the bandintensities were quantified by PC-based image analysis (Image AnalysisInc., Ontario, Canada).

Affinity Precipitation

Affinity precipitation with GST-PBD was performed using an assay basedon the method of Benard et al [22]. Cells were lysed in Mg²⁺ lysisbuffer (MLB), that was provided by the assay kit (UBI, Lake Placid,N.Y.), were mixed with 8 g GST-PBD bound to glutathione-Agarose andincubated for 1 h at 4° C. Pre-cipitates were washed three times withMLB and suspended in Laemmli's sample buffer. Proteins were separated by11% SDS-PAGE, transferred onto nitrocellulose membrane, and blotted withanti-Cdc42 or anti-Rac antibody.

In Vivo Effect of Testosterone-BSA in Nude Mice

Nude mice were injected in the back with 5×10⁶ LNCaP cells diluted inMatrigel® (Sigma, St Louis, Mo.) in a total volume of 0.1 ml. After 4weeks, macroscopical tumors were developed, and treatment was initiated,as follows: Drugs, diluted in PBS were injected intraperitoneally 3times per week, in a total volume of 0.5 ml. Animals were divided infour groups: The first group received 5×10⁻⁶ M BSA. The second group wasinjected with 5×10⁻⁶ M testosterone-BSA conjugate. The third group wasinjected with 10 mg/ml Taxol®, while in the fourth group a combinationof testosterone-BSA and Taxol® was introduced. Tumors were measuredafter four weeks of treatment. Tumors were excised, measured, weightedand send to a pathologist for further analysis.

Results Membrane Androgen Binding Sites on the Human Prostate CancerCell Line LNCaP

Membranes, prepared from cultures of LNCaP cells were incubated withdifferent concentrations of [³H] testosterone (ranging 2-50 nM) without(total binding) or with (non-specific binding) a 1000-fold molar excessof unlabelled androgen (DHT). After overnight incubation at 4° C.,membrane-bound radioactivity was separated and counted. It was found, aspresented in FIG. 1A, that [³H] testosterone, ranging from 1 to 50 nM,induces a specific saturable binding. Scatchard analysis of the results(FIG. 5A insert) revealed a high binding affinity for testosterone(K_(D) 10.9 nM) and a number of binding sites of 144.3 fmoles/mgprotein, corresponding to an approximate number of 13340 sites/cell.

The androgen selectivity of this membrane-binding component was verifiedby competition displacement experiments. Membranes were incubated with[³H] testosterone in the presence of varying concentrations of DHT orother steroids (10⁻¹²-10⁻⁶ M). As shown in FIG. 1B, DHT produced adisplacement of radiolabeled testosterone. In contrast, estradiol andprogesterone displaced radiolabelled testosterone with a significantlower affinity (10⁴- and 10²-fold respectively) confirming the androgenselectivity of the identified membrane-binding site.

The presence of membrane testosterone receptors' was equally identifiedusing the testosterone analogtestosterone3-(O-carboxymethyl)oxime-BSA-FITC, obtained from Sigma (StLouis, Mo.). This analog is not capable to penetrate the cells, becauseof a covalent attachment of the steroid with BSA. As shown in FIG. 2,left panel, a specific membrane binding of testosterone-BSA was found,by flow cytometry, on membranes of LNCaP cells. The association oftestosterone-BSA with membrane receptors was observed at 1 min, wasmaximal after 10 min, and remained unchanged after 30 min of incubation.The membrane binding was equally verified by confocal laser microscopy,as shown in the right panel of FIG. 2. As shown, only membrane stainingwas found by the use of the testosterone-BSA conjugate, ruling out thehypothesis of a possible internalization of the compound.

From these experiments it was concluded that prostate cancer cellspossess specific, high affinity membrane binding sites, which areselective for androgens.

Identification of Membrane Testosterone Receptors in Specimens ofProstate Cancer

In a series of 14 prostate cancer specimens, 10 transurethral resectionsfor benign prostate hyperplasia (BPH), and 8 microscopically verifiednon-malignant specimens from the same cases, we have prepared epithelialcell specimens. Cells were immunostained with monoclonal antibodies tovimentine, cytokeratine and PSA, to evidence stromal, and normal ormalignant epithelial cells respectively. It was verified that epithelialcells accounted for more than 85% of total cells, in all studiedspecimens. Cells were incubated for 10 min with testosterone-BSA andassayed by flow cytometry. As shown in FIG. 3, membrane testosteronebinding was very low in cases of BPH, while a high binding was found inall cases with cancer. In this respect, membrane testosterone receptorscan fully discriminate malignant from benign cases of prostate tumors.

The above discrimination was apparent also by fluorescent staining ofprostate epithelial cells in touch preparations (FIG. 4, upper panel).Indeed, after surgery, gross identified malignant lesions of thesurgical preparations were touched on SuperFrost/Plus slides, adheredcells were stained with testosterone-BSA-FITC, and immediately analysedin a fluorescent microscope. As shown, only malignant epithelial cellswere stained, while BPH epithelial cells presented a very lowfluorescence. Finally, as shown in the lower panel of FIG. 4, membranetestosterone receptors can be identified in routine histological slides,from formalin-fixed, paraffin-embedded cases of prostate cancer. It isinteresting to note further, that testosterone membrane staining canidentify cases of intraepithelial neoplasia specifically.

From the above results, it becomes apparent that testosterone membranereceptors are a specific and selective element of prostate cancer.

Identification of Membrane Steroid Receptors in Breast Cancer Specimens

Estrogen-progesterone- and androgen-membrane binding was assayed insteroid receptor positive and negative tumors, as assayed byimmunocytochemistry. Typical results are presented in FIG. 5. As shown,regardless of the state of intracellular steroid receptors,BSA-conjugated steroids identify components in histologicalpreparations. Androgen receptors are present in low concentrations inthese breast tumors. In contrast, estradiol-BSA and progesterone-BSAidentify pericellular components in tumoral cells in the breast. This ismore obvious in ER/PR negative tumors, in which there is no interactionwith intracellular receptors. Indeed, in ER/PR positive cases, as thereis a cellular damage, during slide preparation, these exist, in somecases, a diffuse pattern of staining, which, in some cases can not beattributed to a pericellular, intracellular, or nuclear binding.

Identification of Membrane Steroid Receptors in HematologicalMalignancies

In normal blood white blood cells (WBC) we have identified testosteronemembrane binding (performing routine flow cytometric assays). Thedistribution of testosterone-positive cells are shown in Table 1.

TABLE 1 Distribution of membrane testosterone positive (Testo+) cells indifferent groups of WBC in 20 health blood donors. Lymphocytes MonocytesPolymorphonuclear % of total 35.6 ± 1.03 7.7 ± 0.41 56.1 ± 1.18 WBCTestosterone+ 8.9 2.2 11.6 % Testo+ in 23 28 20 category

As shown, membrane testosterone receptors are expressed in all threeclasses of WBC. It is interesting that monocytes express, in higherpercentages these sites.

The analysis of testosterone membrane receptor in subclasses oflymphocytes is expressed in Table 2.

TABLE 2 Distribution of membrane testosterone positive cells indifferent categories of lymphocytes. T cells were assayed by the assayof CD3 marker, B by the expression of CD19, and NK cells by theexpression of CD56 lymphocyte antigen. Coexpression of the above marker(marked by a PE-labeled monoclonal antibody) with testosterone-BSA-FITCwas used for the detection of the testosterone positive subset of cells.T B NK % of total 76 14 9.7 Lymphocytes Testosterone+ 18 4.25 3.4 %Testo+ in 23 30 34 category

As shown, B-lymphocytes and NK cells express preferentially thetestosterone receptor, as compared to T-lymphocytes. In addition,further analysis of Tcells, showed an equal distribution in CD4 and CD8positive lymphocytes.

In four cases of malignancies, the distribution of testosterone membranereceptor is shown in Table 3.

TABLE 3 Detection of membrane testosterone binding in four cases ofhematological malignancies. The mean of the normal controls is given forcomparison. Lymphocytes Monocytes Polymorphonuclear Diagnosis % % Testo+% % Testo+ % % Testo+ ALL remission 19 24 8 25 73 19 ALL 81 16 1 25 1814 AML Remission 32 24 10 35 58 27 Malignant 70 19 7 34 23 16 LymphomaNormal 36 25 8 28 57 21

As shown, membrane testosterone binding was found decreased inlymphocytes in ALL, while it returns to the levels of normal controls inremission. In contrast, in malignant lymphma and AML, increasedtestosterone membrane binding is found in monocytes, while in ALL andthe case of lymphoma studied, polymorphonuclear membrane testosteronereceptors are found to be decreased.

In Table 4, it is presented the distribution of testosterone receptorsin different subclasses of lymphocytes. As shown, membrane testosteronereceptors are equally low in all three subsets of lymphocytes, in thecase of ALL, returning to normal values after remission. The same resultis also found in the case of AML.

TABLE 4 Distribution of membrane testosterone receptors, in four casesof hematological malignancies. Results obtained in normal blood donorsare given for comparison. T B NK Diagnosis % % Testo+ % % Testo+ % %Testo+ ALL remission 85 20 35 6 10 3 ALL 64 9 5 2 2 0.2 AML Remission 7313 5 1 15 5 Malignant Lymphoma 90 17 13 4 5 1 Normal 76 18 14 4 10 3

It is interesting to note that the distribution of testosterone membranesites shows a differential distribution in normal and leukemic cells. Inaddition, as shown in FIG. 6, bone marrow stem cells (both CD34 andAC133 positive) express membrane binding sites for all three steroidstested (estrogen, progesterone and androgen). It is therefore possiblethat the expression of non-mature lymphoid cells account for thedifferential expression of membrane testosterone receptors, andtherefore, the invention may also be used for the detection andtreatment of hematological malignancies.

Interaction of Membrane Steroid Receptors with Actin Cytoskeleton

FIG. 7 shows the effect of action of testosterone-BSA conjugate to theactin cytoskeleton of LNCaP human prostate cancer cells, assayed byconfocal laser scanning microscopy. As depicted, 10 minutes aftertestosterone application, a profound modification of the cytoskeletonoccurs. Actin filaments are redistributed at the periphery of the cell,while, as presented at the lower panel of FIG. 7, the significantdecrease of the ratio of soluble (monomeric) to insoluble (polymerised)actin indicates that profound alterations of the actin cytoskeletonoccur, in favor of a polymerisation process.

Further work revealed that testosterone receptors located on cellmembranes of LNCaP cells activate key signalling molecules in ahierarchy of FAK→PI-3 kinase→Cdc42/Rac1→actin reorganization. The factthat testosterone was less active than testosterone-BSA conjugatefurther indicates that this signalling cascade might be specific of theactivation of testosterone membrane binding sites. These resultsoutline, for the first time, a signal transduction pathway that wastriggered by membrane testosterone receptors in prostate cancer cellsand leads to actin reorganization.

Long Term (24 hours) Incubation with Testosterone-BSA Decrease CellProliferation of Cancer Cells

In view of the above results, we incubated prostate cancer LNCaP cellswith testosterone-BSA alone (10⁻⁷ M) or together with Taxol® (10⁻⁸ M)for 24 hours. As shown in FIG. 8A, a 50% decrease of cells incubatedwith testosterone −BSA alone was found. In addition, a potentiation ofthe action of Taxol® by ˜7% was also found. If medium was replaced afterthis 24 hours incubation, and cells were provided with fresh mediumwithout any added substance and let to stay for additional 48 hours (acondition mimicking the weekly administration of low doses of Taxol® inclinic), cells recover partially (FIG. 8B). In this case, the action oftestosterone-BSA is more potent than that of Taxol®. This effect isdose-related to the testosterone-BSA, as shown in FIG. 8C, indicating anadditive effect of testosterone-BSA with Taxol®.

In Vivo Effects of Testosterone-BSA in Nude Mice.

All mice supported very well the treatment with testosterone-BSA, at theconcentrations used. As presented in FIG. 9, testosterone-BSA induced atime-dependent decrease in tumor mass by 53%. Addition of Taxol®produces a dramatic decrease of tumor weight by 77%. The experiment wasstopped at four weeks, while testosterone-BSA did not reach a plateau.Therefore it is possible that the decrease if tumor mass could begreater for longer periods of treatment. In addition, apoptosis wasobserved histologically in all tumors studied.

REFERENCES

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1-25. (canceled)
 26. A method for treating a solid or hematologicalcancer, the method comprising administering to a patient in need thereofa composition comprising: one or more androgen steroids, wherein theandrogen steroid is covalently attached to a mammalian protein excludingan antibody and the cancer comprises membrane androgen receptor-bearingcells.
 27. The method of claim 26, wherein the solid cancer is one ofprostate adenocarcinoma (hormone sensitive or resistant) and metastasesthereof, breast cancer and metastases thereof, pheochromocytomas andmetastases thereof, bone tumor and metastases thereof and brain tumor(neuroblastomas).
 28. The method of claim 26, wherein the hematologicalcancer is one of acute or chronic myeloid leukemia, acute or chroniclymphoid leukemia and a lymphoma (B or T).
 29. The method of claim 26,wherein the androgen is testosterone.
 30. The method of claim 26,wherein the mammalian protein is a recombinant or isolated natural serumalbumin.
 31. The method of claim 26, wherein the one or more androgensteroids or the mammalian protein is detectably-labeled.
 32. The methodof claim 29, wherein the testosterone is covalently attached to themammalian protein through a carboxy-methyl ether linker.
 33. The methodof claim 32, wherein the linker is covalently attached to thetestosterone at the 3′ position of the steroidal ring.
 34. The method ofclaim 26, the composition further comprises an antiandrogen.
 35. Themethod of claim 34, wherein the antiandrogen is present in about a10-fold molar excess relative to the molar amount of the one or moreandrogen steroids.
 36. The method of claim 26, wherein theadministration of the composition is parenteral, percutaneous orintravenous.
 37. The method of claim 26, wherein the composition isadministered at least once daily.
 38. The method of claim 26, whereinthe method further comprises the step of administering an antiandrogento the patient.
 39. The method of claim 26, wherein the method furthercomprises the step of decreasing solid cancer mass in the patient.
 40. Amethod for treating a solid or hematological cancer, the methodcomprising administering to a patient in need thereof a compositioncomprising one or more androgen steroids, wherein the androgen steroidis covalently attached to a mammalian protein excluding an antibodyselected from the group consisting of a globular protein, a plasmaprotein and albumin; and wherein the cancer comprises membrane androgenreceptor-bearing cells.
 41. The method of claim 40, wherein the solidcancer is one of prostate adenocarcinoma (hormone sensitive orresistant) and metastases thereof, breast cancer and metastases thereof,pheochromocytomas and metastases thereof, bone tumor and metastasesthereof and brain tumor (neuroblastomas).
 42. The method of claim 40,wherein the haematological cancer is one of acute or chronic myeloidleukemia, acute or chronic lymphoid leukemia and a lymphoma (B or T).43. The method of claim 40, wherein the androgen steroid istestosterone.
 44. The method of claim 40, wherein the mammalian proteinis a recombinant or isolated natural serum albumin.
 45. The method ofclaim 40, wherein the one or more androgen steroids or the mammalianprotein is detectably-labeled.
 46. The method of claim 43, wherein thetestosterone is covalently attached to the mammalian protein through acarboxy-methyl ether linker.
 47. The method of claim 46, wherein thelinker is covalently attached to the testosterone at the 3′ position ofthe steroidal ring.
 48. The method of claim 40, wherein the compositionis administered at least once daily.
 49. The method of claim 40, whereinthe method further comprises the step of decreasing solid cancer mass inthe patient.
 50. A method for treating a solid cancer, the methodcomprising administering to a patient in need thereof a compositioncomprising: (a) one or more androgen steroids, wherein the androgensteroid is covalently attached to a mammalian protein excluding anantibody; and wherein the solid cancer is prostate adenocarcinoma(hormone sensitive or resistant) and metastases thereof, breast cancerand metastases thereof, pheochromocytomas and metastases thereof, bonetumor and metastases thereof or brain tumor (neuroblastomas) and whereinthe cancer comprises membrane androgen receptor-bearing cells.
 51. Amethod for treating a solid cancer, the method comprising administeringto a patient in need thereof a composition comprising: (a) one or moreandrogen steroids, wherein the androgen steroid is covalently attachedto a mammalian protein excluding an antibody selected from the groupconsisting of a globular protein, a plasma protein, and albumin; andwherein the solid cancer is prostate adenocarcinoma (hormone sensitiveor resistant) and metastases thereof, breast cancer and metastasesthereof, pheochromocytomas and metastases thereof, bone tumor andmetastases thereof and brain tumor (neuroblastomas) and wherein thecancer comprises membrane androgen receptor-bearing cells.
 52. Themethod of any one of claims 26, 40, 50 or 51, wherein the solid canceris breast cancer.